Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing otitis media in children. Lipooligosaccharide (LOS), one of the most important surface antigens in NTHi, is immunogenic and a potential target of bactericidal antibodies during Haemophilus otitis media. We have prepared detoxified LOS-protein conjugates as a possible vaccine candidate to induce a long-term immune response for the prevention of NTHi otitis media in young children. In order to characterize biologically important epitopes of NTHi, twenty- one mouse MAbs against strain 9274 were prepared. The isotypes, the specificity and the functions of these MAbs were characterized. The results showed that two of these MAbs are IgG3 and the others are IgM. Two of these MAbs are against outer membrane proteins and the others are against LOS. Interestingly, ten of the MAbs showed bactericidal activities and some of them showed opsonophagocytic killing with human polymorphonuclear leukocytes. These results indicate NTHi LOS contains protective epitopes which can be used as a vaccine component. Further investigation will be needed to identify these functional epitopes.